All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.
Current strategies for targeting therapy to tumors include antibody-targeted chemotherapy agents (i.e., immunoconjugates), targeted toxins, signal-blocking antibodies, and antibody-targeted liposomes (i.e., immunoliposomes). In fact, HER2+ breast tumors, which over-express subunit 2 of the human epidermal growth factor receptor (HER), comprise a significant subset of breast cancers that are recalcitrant to standard methods of treatment, and predict a high mortality. The abnormally high level of HER on the surface of these tumor cells may enable targeted therapeutics to home in on these cells, thus making HER2+ tumors ideal candidates for targeted therapy. However, the aforementioned therapies are problematic—with respect to HER2+ breast tumors and other forms of cancer—because they require chemical modification which may be costly and can impair activity of the drug and/or the carrier; may use recombinant antibodies that can lose structure in physiological conditions and thus result in impaired targeting activity; are not able to penetrate into the cell; focus on the need to modulate receptor signaling which can be impaired in tumor cells; and have off-target effects such as heart toxicity.
Immunoconjugate therapies rely on the chemical coupling of single-chain antibodies to drugs, whereby the antibody directs the drug to specific cells by recognizing certain cell surface proteins or receptors [K. A. Chester et al., Disease Markers, 16:53-62 (2000); A. E. Frankel et al., Clin. Cancer Res., 6:326-334 (2000)]. Studies have shown, however, that such antibodies can unfold and aggregate at physiological temperature, which would impede binding to target cells, and result in low therapeutic efficacy [R. Glockshuber et al., Biochem, 29:1362-1367 (1990); M. Schmidt et al., Oncogene, 18:1711-1721 (1999)]. Moreover, covalent linkage of drug to carrier can impair the activity of both molecules, as well as entail high production cost.
In contrast, studies in connection with one embodiment of the present invention show that an exemplary targeted carrier protein, HerPBK10, retains receptor targeting under physiological conditions in the presence of serum, indicating that the targeted carrier does not unfold or lose receptor binding [H. Agadjanian et al., Pharm Res., 23:367-377 (2006)]. Furthermore, the present invention is engineered so that the drug self-assembles with the carrier molecule and thus does not require chemical modifications to covalently link the molecules together. This non-covalent assembly thus allows both the targeted carrier and drug to remain unmodified and thus preserve structure and activity. Finally, recombinant proteins (such as HerPBK10) can be produced in bulk quantities by large-scale fermentation for a lower cost compared to monoclonal antibody generation and production.
An alternative approach to tumor targeting has been the development of toxic proteins, such as plant or bacterial toxins, that are modified by appendage to a targeting peptide (or ligand) [A. E. Frankel et al., Clin. Cancer Res., 6:326-334 (2000)]. Such proteins are produced by recombinant methods (i.e., genes are engineered to produce the proteins in a cell system, from which the proteins can then be isolated), and the resulting protein is a fusion of the toxin to the ligand. While fusion toxin proteins can be produced by large scale fermentation as a more efficient and lower cost alternative to antibody generation, the toxin requires special processing to be active, thus limiting potency [M. Schmidt et al., Oncogene, 18:1711-1721 (1999); M. Jeschke et al., Intl. J. Cancer, 60:730-739 (1995)]. For example, the activity of recombinant diptheria toxin transmembrane domain, used to enhance non-viral gene transfer, is reduced by as much as 75% when fused to a foreign peptide, indicating that appending a peptide to a toxin disables toxin activity [K. J. Fisher and J. M. Wilson, Biochem. J., 321:49-58 (1997)]. Thus, delivery by non-covalent means (i.e., self-assembly), which is a feature of the present invention, is believed to be advantageous in terms of retaining drug potency.
Antibodies directed at the extracellular domain of HER2 have been used to target drug complexes to the HER2 subunit, but have not necessarily induced internalization of the drug complex; thus limiting potency [D. Goren et al., Br. J. Cancer, 74:1749-1756 (1996)]. Such findings illustrate that targeting is not enough, as a lack of targeted uptake can limit efficacy. Alternatively, signal blocking antibodies have been developed to inhibit the proliferative signal transduced through overexpression and high cell surface display of HER2 [J. Baselga et al., J. Clin. Oncol., 14:737-744 (1996); M. A. Cobleigh et al., Proc. Am. Soc. Clin. Oncol., 17:97a (1998)]. One currently used targeted therapy, trastuzumab (Herceptin), an antibody directed against the HER2 subunit, blocks normal signaling but has been ineffective in about 70% of treated patients, possibly due to aberrant intracellular pathways in tumor cells that may not respond to signal inhibition [C. L. Vogel et al., “J. Clin. Oncol., 20:719-726 (2002); T. Kute et al., Cytometry, A57:86-93 (2004)]. Furthermore, an ongoing concern with trastuzumab is the exquisite sensitivity of heart tissue to HER2 signal inhibition, which is further exacerbated by anthracycline chemotherapy agents [D. J. Slamon et al., N. Engl. J. Med., 344:783-792 (2001)]. In one embodiment, the approach of the present invention instead takes advantage of the binding interaction of the natural ligand for HER, which has a greatly increased ligand affinity when HER2 is overexpressed. It is believed that this is likely to translate to lower, and thus safer, doses of drug when targeted. Accordingly, the targeting approach of the present invention should avoid binding to tissues displaying low to normal receptor subunit levels but exhibit preferential binding to HER2+ tumor cells. Moreover, the inventor has shown that the receptor binding domain of heregulin that is incorporated into HerPBK10 induces rapid internalization after binding to the heregulin receptor [L. K. Medina-Kauwe et al., BioTechniques, 29:602-609 (2000); L. K. Medina-Kauwe and X. Chen, Vitamins and Hormones, Elsevier Science, G. Litwack (Ed.), San Diego, 81-95 (2002)]; enabling uptake of DNA [L. K. Medina-Kauwe et al., Gene Ther., 8:1753-1761 (2001)] and fluorescent compounds [H. Agadjanian et al., Pharm Res., 23:367-377 (2006)]. The inventive approach, therefore, circumvents the need to modulate receptor signaling, by exploiting the rapid receptor endocytosis induced by ligand binding and the cytosolic penetration features of viral capsid protein to directly transport drugs into the cell and induce cytotoxicity from within.
While targeted uptake facilitates drug entry into target cells, the intracellular disposition of the drug can still affect potency. Targeting antibodies delivering covalently linked drugs can be trafficked to lysosomes, thus sequestering the drug from subcellular targets and limiting potency. Approaches to circumventing this include linking the drug to a targeting antibody via an acid labile bond to facilitate release into the endocytic compartment [P. A. Trail et al., Cancer Immunol Immunother., 52:328-337 (2003); P. A. Trail et al., Cancer Res., 52:5693-5700 (1992); P. A. Trail et al., Science, 261:212-215 (1993); G. R. Braslawsky et al., Cancer Res., 50:6608-6614 (1990)]. However, bond instability can reduce in vivo potency, likely by causing premature drug release and thus delivery to non-target tissue. In contrast, the endosomal disruption feature of one embodiment of the present invention has the advantage of endosomal escape, thus facilitating release of the drug into the cell cytoplasm and access to intracellular targets, which can include passengering of nuclear targeted molecules and passage through nuclear pores.
Immunoliposomes, carrying doxorubicin (“Dox”) and targeted to HER2, have been developed and can accumulate in tumor tissue in animal models [J. W. Park et al., Clin. Cancer Res., 8:1172-1181 (2002)], likely due to the leaky tumor vasculature D. C. Drummond et al., Pharmacol. Rev., 51:691-743 (1999)]. Release of Dox from these liposomes is thought to occur via the acidic tumor environment, lipase release from dying cells, and enzyme and oxidizing agent release from infiltrating inflammatory cells [G. Minotti et al., Pharmacol. Rev., 56:185-229 (2004)]. These conditions may induce premature drug release and nonspecific delivery, though the accumulation of immunoliposomes at tumor sites may tend to favor drug uptake at the tumor. Studies using the trastuzumab Fab′ fragment for liposome targeting of Dox have demonstrated antitumor efficacy [J. W. Park et al., Clin. Cancer Res., 8:1172-1181 (2002], though the effect on cardiac tissue was not reported in that study. In contrast, for reasons described above, it is believed that in an embodiment, the inventive system yields effective targeted drug delivery due to high affinity receptor-ligand binding and rapid endocytosis coupled with the membrane-penetrating activity of the viral penton base protein to ensure delivery into target cells.